Mouse showing neurofibril change due to senile dementia

ABSTRACT

A transgenic mouse transformed with a DNA encoding a mutant human tau protein, wherein the mouse exhibits neurofibrillary tangles in its brain. The transgenic mouse is useful for the development of the agent for treating neurodegenerative disorders such as Alzheimer&#39;s disease.

This application is a 371 of PCT/JP01/05070 filed Jun. 14, 2001, whichclaims priority from JAPAN 2000-179723 filed Jun. 15, 2000.

TECHNICAL FIELD

The present invention relates to a transgenic mouse that exhibitsneurofibrillary tangles in its brain.

BACKGROUND ART

Alzheimer's disease is specific to human beings. It takes 10 years ormore before its onset so that development of human disease model animalsis desired in order to elucidate the cause of its occurrence or treatit. Alzheimer's disease (also called “Alzheimer type senile dementia”)is macroscopically characterized by atrophy of the brain, in particular,atrophy of temporal lobe while under microscope, it shows pathologicalchanges characteristic mainly of neurofibrillary tangles and senileplaques. Neurofibrillary tangles (NFT) are neuronal inclusions composedmainly of phosphorylated tau protein. Neurofibrillary tangles areobserved in sites where neurons are abolished, besides in Alzheimer'sdisease, in neurodegenerative disorders with dementia (frontotemporaltype dementia Parkinsonism-17 (FTDP-17) (Poorkaj, P. et al. Tau is acandidate gene for chromosome 17 frontotemporal dementia. Ann. Neurol.43, 815–825 (1998)), Down's syndrome and several other neurodegenerativedisorders). From this it is said that formation of neurofibrillarytangles is a common cascade in all the disorders that involveneurodegeneration. In other words, the formation of neurofibrillarytangles represents a final common pathway that leads to abolition ofneurons in Alzheimer's disease and other neurodegenerative disorderswith dementia (Spillantini, M. G. & Goedert, M. Tau protein pathology inneurodegenerative diseases. Trends Neurosci 21, 428–433 (1998)).Therefore, inhibition of neurofibrillary tangles is considered to be oneof treatments for all the neurodegenerative disorders inclusive ofAlzheimer's disease. However, generation of mice that causeneurofibrillary tangles has hitherto been tried using overexpression ofmutant human amyloid precursor protein (Games, D. et al., Alzheimer-typeneuropathology in transgenic mice overexpressing V717 β-amyloidprecursor protein, Nature 373, 523–527 (1995) and Hsiao, K. et al.,Correlative memory deficits, Aβ elevation, and amyloid plaques intransgenic mice, Science 274, 99–102 (1996)) or human wild-type tauprotein (Tau), and although senile plaques was formed or tau protein wasaccumulated within cells, no neurofibrillary tangle was observed ineither case. That is, although animals that express an amyloid precursorprotein and presenilin were generated as a model animal for Alzheimer'sdisease, they did not exhibit neurofibrillary tangles which representpathological change in dementia although they exhibit senile plaqueslike humans.

Also, it has been tried to generate a transgenic mouse that expresseswild human type tau protein (tau) and three lines of transgenic micehave been reported. In the transgenic mice, (1) 4 repeat tau cDNA drivenby Thy-i promoter (Gotz, J. et al. Somatodendritic localization andhyperphosphorylation of tau protein in transgenic mice expressing thelongest human brain tau isoform. EMBO J. 14, 1304–1313 (1995), (2) 3repeat tau cDNA driven by 3-hydroxy-3-methylglutaryl coenzyme Areductase promoter (Brion, J. P., Tremp, G. & Octave, J. N. Transgenicexpression of the shortest human tau affects its compartmentalizationand its phosphorylation as in the pretangle stage of Alzheimer'sdisease. Am. J. Pathol. 154, 255–270 (1999)), (3) 3 repeat tau cDNAdriven by prion protein promoter (Ishihara, T. et al., Age-dependentemergence and progression of a taupathy in transgenic miceoverexpressing the shortest human tau isoform. Neurone 24, 751–762(1999)) have been respectively used as a transgene.

It has been reported that among the three lines, the neurons in thelines (1) and (2) expressed approximately a 2-fold increase in tau andexhibited pretangle neuropathology while the neurons in the line (3)expressed a 5 to 10-fold increase in tau and the tau inclusions wereobserved in the neurons. However, none of the lines exhibited tauaggregations that showed birefringency after Congo red staining orthioflavin-S reactivity and did show formation of filamentous tauaggregations, i.e., neurofibrillary tangles.

On the other hand, there has been a report that in the mutant tauprotein observed in FTDP-17, valine, which is amino acid at residue 337is substituted with methionine (V337M) (Poorkaj, P. et al., Tau is acandidate gene for chromosome 17 frontotemporal dementia. Ann. Neurol.43, 815–825 (1998)).

However, no report has been made on the generation of a transgenic mousethat expresses mutant human tau.

DISCLOSURE OF THE INVENTION

Thus, since the formation of neurofibrillary tangles is a common cascadefor neurodegenerative disorders, the formation of neurofibrillarytangles is a useful index for the development of future pharmaceuticalsfor intended to treat various neurodegenerative disorders and inhibitionof the formation of neurofibrillary tangles has a potential of directlyleading to the therapy of neurodegenerative disorders. Therefore, atransgenic mouse exhibiting neurofibrillary tangles may provide an idealpreclinical model for the testing of therapeutic drugs for the treatmentof neurodegenerative disorders such as Alzheimer's disease. Accordinglyan object of the present invention is to provide a transgenic mouseuseful for the development of such a therapeutic drug.

To solve the above-mentioned problem, the present inventors have madeintensive investigation and, as a result, they have been found that amouse exhibiting the same neurofibrillary tangles as observed in a humanbrain with Alzheimer's disease can be generated by expression of amutant tau protein which is a cause of familial frontotemporal typedementia, and, based on the findings, they have completed the presentinvention.

Accordingly, the present invention provides a transgenic mousetransformed with a DNA encoding a mutant human tau protein, wherein themouse exhibits neurofibrillary tangles in its brain.

The mutant human tau protein is preferably a mutant tau protein in whichvaline at a position corresponding to residue 337 in SEQ ID NO: 9 issubstituted with an amino acid other than valine. The amino acid otherthan valine is preferably methionine.

Preferably, the transgenic mouse of the present invention is used as amodel of neurodegenerative disorder.

The transgenic mouse of the present invention is preferably onecharacterized in that the neurofibrillary tangles are positive inimmunoreactions using an antibody against a phosphorylated tau protein,an antibody against ubiquitin and an antibody against paired-helicalfilaments, and birefringency is shown after staining with Congo red.

Further, the transgenic mouse of the present invention preferably showsabnormal behavior.

The transgenic mouse of the present invention is preferably one in whichmutant human tau protein is expressed under control of platelet growthfactor β chain promoter.

The present invention also provides a method for screening for an agentfor treating a neurodegenerative disorder, comprising administering acandidate substance to the above-mentioned transgenic mouse exhibitingneurofibrillary tangles, or contacting a candidate substance with atissue or a cell from brain of the transgenic mouse, and determining aneffect of the candidate substance on a property associated with theneurodegenerative disorder in the transgenic mouse or the tissue or thecell thereof, as well as, a method for producing an agent for treatinganeurodegenerative disorder, comprising selecting a candidate substanceby the screening method, and preparing a pharmaceutical compositioncomprising the selected substance.

The transgenic mouse exhibiting neurofibrillary tangles of the presentinvention may play an important role in elucidating the cause ofneurofibrillary tangles and be useful for the development of the agentfor treating all the neurodegenerative disorders exhibitingneurofibrillary tangles including Alzheimer's disease.

The transgenic mouse of the present invention, which exhibitsneurofibrillary tangles, can be used as an ideal preclinic model of aneurodegenerative disorder such as Alzheimer's disease and contributesto elucidation of human neurodegenerative disorders and development oftherapeutic agents therefore.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a map of a mutant human tau-encoding construct used togenerate a transgenic mouse of the present invention.

FIG. 2 shows a photograph showing the results of Southern blot analysisof a DNA fragment introduced into a transgenic mouse by using tau DNAdigested with XhoI and NotI as a probe. Lane 5, Tg216; lane 6, Tg214;and lane 7, Tg212. Lane 1, null transgene copy; lane 2, 1 transgenecopy; lane 3, 5 transgene copies; lane 4, 10 transgene copies; and lane8, 20 transgene copies.

FIG. 3 shows photographs showing the results of Western blot analysis ofthe brain tissue of a transgenic mouse. (a) shows the results obtainedby using an antibody as a probe, which is specific to the mutant humantau. Lane 1, non-transgenic littermate of Tg214; Lane 2, non-transgeniclittermate of Tg216; lane 3, Tg212; lane 4, Tg214; and lane 5, Tg216.(b) shows the results obtained by using an anti-tau antibody, JM, as aprobe, which recognizes both endogenous and mutant human tau. Lanes 1 to5 are respectively given same assignments as in (a) above.

FIG. 4 shows photomicrographs showing the expression of mutant humantau. (a) shows expression in the cerebral cortex of Tg216 (Scale bar: 50μm). (b) shows expression in hippocampus of Tg214 (scale bar: 50 μm)

FIG. 5 shows photomicrographs of Congo red stained sections of braintissue (scale bar: 10 μm). (a) is the hippocampus of Tg214 (10 monthsold). (b) is the hippocampus non-transgenic littermate of Tg214. (c) isthe cerebral cortex of Tg216 (10 months old). (d) is the cerebral cortexof human brain with Alzheimer's diseases.

BEST MODE FOR CARRYING OUT THE INVENTION

<1> DNA Encoding Mutant Human Tau Protein

Human tau protein is known to include several isoforms. The nucleotidesequence of MRNA encoding one of isoforms of the human tau protein isregistered under GenBank/EMBL/DDBJ accession No. J03778. Because thehuman tau protein includes isoforms, the position of amino acid isherein indicated by the number of the corresponding position in theamino acid sequence shown in SEQ ID NO: 9.

The DNA encoding mutant human tau protein used in the present inventionencodes mutant human tau protein having mutation by which a mousetransformed with the DNA exhibits neurofibrillary tangles in its brain.The mutant human tau protein is preferably one observed in the diseaseexhibiting neurofibrillary tangles.

The mutant human tau protein having such a mutation includes one havingan amino acid sequence in which the amino acid residue at 337, which isvaline in the tau protein of normal person, is substituted with an aminoacid other than valine (preferably methonine) and one having an aminoacid sequence in which the amino acid residue at 406, which is argininein the tau protein of normal person, is substituted with an amino acidother than arginine (preferably tryptophan).

Since it has been known that some amino acids have similar properties tothose of others and that generally the amino acid sequence of a proteinmay contain a part that has no influence on the activity of the protein,it is expected that mutant tau protein may have mutation that has noinfluence on the activity that results in the neurofibrillary tangles inaddition to the mutation exhibiting neurofibrillary tangles in the brainof a mouse transformed with a DNA encoding the mutant tau protein. TheDNA encoding an amino acid sequence may include different nucleotidesequences due to degeneration of genetic codons.

Therefore, the nucleotide sequence of DNA encoding, for example, themutant tau protein in which valine at 337 is substituted with methionine(hereinafter, also referred to as “V337M human tau”) may havepolymorphism such as substitution, deletion, or insertion of one or morenucleotides as long as V337M human tau is expressed and the transgenicmouse exhibits neurofibrillary tangles.

<2> Transgenic Mouse Exhibiting Neurofibrillary Tangles of the PresentInvention

The transgenic mouse of the present invention is characterized byexhibiting neurofibrillary tangles similar to those observed in thehuman brain with Alzheimer's disease. The neurofibrillary tangles (tauaggregation) in the brain of transgenic mouse are those satisfyingpathological changes and histological characteristics characteristicallyobserved in the brain of patients with Alzheimer's disease. Humanneurofibrillary tangles refer to argento-philic nerve structuresaccumulated in neurons and are composed of a large number of fibrilsrunning parallel to each other. A single fibril is called paired helicalfilaments (PHF). The PHF is a unique fibril that has constrictions at aninterval of about 80 nm. Its basic backbone is composed ofhyperphosphorylated tau. It revealed that this tau is ubiquitinated whensubjected to processing.

The phrase “exhibits neurofibrillary tangles” as used herein means thatin microscopic observations, neurofibrillary tangles are observed in thebrain of the transgenic mouse. More specifically, one or moreimmunoreactions using specific antibodies against phosphorylated tau,ubiquitin and PHF, respectively, are positive or birefringency is shownafter Congo red staining as in human neurofibrillary tangles.Preferably, the phrase means that all the immunoreactions usingrespective specific antibodies against the phosphorylated tau, ubiquitinand PHF are positive and birefringency is shown after Congo redstaining.

Further, the transgenic mouse of the present invention preferably ischaracterized in that it shows an abnormal behavior such as antianxiety.The abnormal behavior means abnormality compared with a control animal.For example, the transgenic mouse stays on an open arm in an elevatedmaze for a significantly longer time than a control animal (usually,mice are apt to dislike approaching an open arm). Usually, the controlanimal in the maze acclimates within 2 minutes to cause a reduction inthe amount of behavior. However, the transgenic mouse does not causethis.

Furthermore, human neurofibrillary tangles are present as bound with RNAand form a β-sheet structure and hence it is preferable that the β-sheetstructure be observed in the neurons in the brain of the transgenicmouse by thioflavin staining, and it is also preferable that theneurofibrillary tangles be in a state where they are bound with RNA.

Specific examples of the antibodies for use in immunoreactions usingspecific antibodies against phosphorylated tau, ubiquitin and PHF,respectively, include PS199 as a phosphorylation dependent anti-tauantibody (Michel, G. et al., Characterisation of tau phosphorylation inglycogen synthase kinase-3β and cyclin dependent kinase-5 activator(p23) transfected cells. Biochimica et Biophysica Acta 1380, 177–182(1998)), AT8 (Biernat, J. et al. The switch of tau protein to anAlzheimer-like state includes the phosphorylation of two serine-prolinemotifs upstream of the microtubule binding region. EMBO J. 11, 1593–1597(1997)) and the like. The antibody that recognizes the structure of PHFincludes an antibody specific for conformational epitope found in PHF,Alz50. For recognizing ubiquitin, anti-ubiquitin antibody may be used.Monoclonal antibodies to the above proteins can be prepared, forexample, by the Cambell method using the above-mentioned proteins fixedwith formalin (MONOCLONAL ANTIBODY TECHNOLOGY, Elstevier SciencePublishers, Amsterdam, 1984) as antigen.

The methods for measuring the above-mentioned properties ofneurofibrillary tangles are described in <4> below.

<3> Generation of Transgenic Mouse

The transgenic mouse of the present invention can be generated asfollows.

(1) Preparation of Mutant Human Tau Protein (Mutant Human Tau) DNAStructure

The structure of human tau DNA is known as described above. Human tauDNA can be obtained by a method for synthesizing its entire sequencebased on a known amino acid sequence or nucleotide sequence(GenBank/EMBL/DDBJ accession No. J03778, etc.), a method using PCR withprimers synthesized based on a known amino acid sequence or nucleotidesequence, or the like method. The human tau DNA may have intron.

The mutant human tau DNA can be prepared, for example, by introducingmutation by site-specific mutagenesis (for example, PCR-basedsite-specific mutagenesis) to a known human tau DNA.

When the mutant human tau is V337V human tau, specific examples ofprimers for the PCR include those having nucleotide sequences shown inSEQ ID NOS: 3 to 8.

The total sequence of the prepared DNA (about 1.4 kb) can be confirmedby a DNA sequencer and the like (for example, ABI PRISM 377 DNAsequencer and the like).

(2) Transgenic Mouse

The transgenic mouse having DNA encoding the mutant tau protein andexhibiting neurofibrillary tangles can be obtained by introducing theDNA into a suitable host to transform it. The host is not particularlylimited and usually used mice can be used.

The transformation, that is, introduction of DNA into a mouse, can beperformed by usual methods used for a mouse, for example, amicroinjection method. More specifically, DNA encoding the mutant tauprotein (transgene) is introduced into an ovum by a microinjectionmethod and the injected ovum is transplanted to a foster mother, andintroduction of the gene to the chromosome of mouse is confirmed usingthe tail DNA of the borne littermate. Thus, a transgenic mouse can beobtained. Since the amount of mutant tau may vary among mouse lines, andamong mice even within the same transgenic line, it is preferable thattwo or more mouse lines be used in generating transgenic mice.

The transgene can be constructed by introducing a region that isnecessary for expressing neurofibrillary tangles into the mutant humantau DNA.

The region necessary for expressing neurofibrillary tangles includes apromoter to be added to the 5′-terminal, a poly-A signal to be added tothe 3′-terminal and the like. The region necessary for expressingneurofibillary tangles may be added via intron. The promoter forexpressing the mutant human tau DNA sequence is not particularly limitedbut promoters usually used when expressing genes in a mouse may be used.As such a promoter, platelet derived growth factor (PDGF)-β chainpromoter is preferably used. The PDGF-β chain promoter can be obtainedfrom a human genome library.

It is preferred to attach an epitope tag to the sequence of human tauprotein near its N-terminal and/or C-terminal in order to distinguishthe human tau protein from endogenous proteins since the human tauprotein exists also as an endogenous protein, or to immunologicallyconfirm expression of the mutant human tau. Examples of the tag epitopenear the N-terminal and/or C-terminal include, for example, myc aminoacid sequence (SEQ ID NO: 1) and FLAG amino acid sequence (SEQ ID NO: 2)(FIG. 1).

Whether a transgenic mouse is generated or not by microinjection can beconfirmed by a genetic technique, e.g., hybridization method such as aSouthern blot method using ³²P-labeled human tau probe (Southern, E. M.,J. Mol. Microbiol. 98, 503, 1975). Also, the expression of mutant tauprotein in the transgenic mice can be immunologically confirmed by theWestern blot analysis by Towbin, H. et al. (Proc. Natl. Acad. Sci. USA,76, 4350, 1979). Further, a transgene-derived mutant tau protein can berecognized by a specific anti-tau antibody.

In the case where the transgene encodes mutant human tau labeled byepitope tags near N-terminal and/or C-terminal, the expression of taucan be confirmed by using an anti-protein antibody to the terminal tag.Also, in this case, the mutant tau protein is greater than theendogenous tau, which has a size of about 50 kD, though depending on thesize of the attached epitope and the like (in Examples, the mutant tauprotein is of a size of about 70 kD), so that it can be distinguishedfrom the endogenous tau.

Specifically, whether the transformed mutant tau protein is expressed ornot can be confirmed, for example, as follows. First, a transformedtransgenic mouse is sacrificed, the mouse brain is homogenised in areassembly buffer (RAB), and tau protein is recovered as heat-stablefractions, which then are separated on SDS-PAGE gel, and the separatedprotein is transferred on a PVDF membrane. Then using an antibody suchas the above-mentioned anti-tau antibody, specific immunoreaction isperformed. If the result of immunoreaction is positive, the expressionof mutant tau protein is confirmed.

<4> Immunohistochemical and Histochemical Studies

(1) Localization of Mutant Tau by Immunostaining

The localization of mutant tau in the brain of a transgenic mouse can bedetermined by immunostaining with an anti-tau antibody, an antibodyagainst a terminal tag epitope (for example, anti-myc antibody) or thelike. Specifically, neurons (cerebral cortex and hippocampal region) inthe brain section of a transgenic mouse are immunostained with ananti-tau antibody (for example, JM described hereinbelow), an antibodyagainst a terminal epitope tag (for example, anti-myc antibody) or thelike and the localization is confirmed under a microscope.

(2) Confirmation of Neurofibrillary Tangles by Histochemical Tests

Whether or not the transgenic mouse of the present invention exhibitsneurofibrillary tangles can be confirmed as follows.

Neurons that exhibit neurofibrillary tangles (NFT) behavior generallyobserved in human neurodegenerative disorders such as Alzheimer'sdisease and frontotemporal dementia Parkinson syndrome-17 areirregularly shaped under microscopic observation and clearly distinctfrom other neurons. As a result, when irregularly shaped neurons ormorphology resembling such neurons are observed under a light microscopeor electron microscope, the existence of cells with mutant tau can bepresumed.

Further, NFT are composed of a number of fibrils running parallel toeach other and a single fibril is called PHF. The basic backbone of PHFis composed of hyperphosphorylated tau that is ubiquitinated and forms aβ-sheet structure. As a result, when a change in which argento-philicfibrillary structure is accumulated in neurons, the neurons are presumedto show immunoreactivity for an antibody against ubiquitinated andphosphorylated tau.

Accordingly, NFT or its resembling morphology can be confirmed byperforming histochemical test used for identifying NFT in humanneurodegenerative disorders. The histochemical tests for detecting NFTinclude immunostaining a brain section from the transgenic mouse with aphosphorylation dependent anti-tau antibody (for example, PS199, AT8,etc.), an anti-ubiquitin antibody, an antibody specific for aconformational epitope of paired helical filaments (PHF) (for example,Alz50, etc.) or the like. And, whether or not a paired helical filament(PHF) epitope or the like observed in neurodegenerative disorders isformed in neurons with the mutant tau can be confirmed by measuring theabove-mentioned immunoreactivity of the brain section. Further, ifmicroscopic observation reveals the existence of linear microtubules ina portion corresponding to PHF associated with neurofibrillary tangles(NFT) in the neurons obtained from the transgenic mouse, it can bepresumed that neurofibrillary tangles are formed.

(3) Confirmation of Whether or not a β-Sheet Structure is Formed byStaining

In the case of neurodegenerative disorders such as Alzheimer's diseaseor Parkinson syndrome, it has been known that in the neurofibrillarytangles (NFT), tau protein is phosphorylated (Morishima-Kawashima, M. etal., Proline-directed and non-proline-directed phosphorylation ofPHF-tau, J. Biol. Chem., 270, 823–829 (1995), Lee, V. M., Balin, B. J.,Otvos, L. Jr. & Trojanowski, J. Q. A68: A major subunit of pairedhelical filaments and derivatized forms of normal Tau., Science, 251,675–678 (1991) and Woloszin, B. L., Pruchnicki, A., Dickson, D. W. &Davies, P., A neuronal antigen in the brains of Alzheimer patients,Science.232, 648–650 (1986)), and ubiquitinated (Mori, H., Kondo, J. &Ihara, Y., Ubiquitin is a component of paired helical filaments inAlzheimer's disease. Science 235, 1641–1644 (1987)) and forms afibrillary structure of a β-sheet configuration. For this reason,whether or not a transgenic mouse exhibits neurofibrillary tangles canbe also confirmed by staining with Cong red or thioflavin-S to see if aβ-sheet structure is formed (upon Congo red staining, the β-sheetstructure emits specific characteristic green color light under apolarizing microscope due to birefringency (Simpson, J., Yates, C. M.,Watts, A. G. & Fink, G., Congo red birefringent structures in thehypothalamus in senile dementia of the Alzheimer type. Neuropathol.Appl. Neurobiol. 14, 381–193 (1988)). That is, by staining a brainsection for mutant tau and then staining it with Congo red orthioflavin-S, the formation of a β-sheet structure and its localizationcan be confirmed. From these observations, it can be presumed that whena mutant human tau (V337M)-expressing transgenic mouse forms tauaggregations forming a β-sheet structure, it exhibits the sameneurofibrillary tangles as those of human.

(4) Counterstaining with Propidium Iodide

It has been confirmed that in the neurofibrillary tangles in the brainof the Alzheimer patient, neurofibrils exist as bound to RNA (Stephen,D. et al., Sequestration of RNA in Alzheimer's disease neurofibrillarytangles and senile plaques. Ann. Neuro. 41, 200–209 (1997) and Nunomura,A. et al., RNA Oxidation is a prominent feature of vulnerable neurons inAlzheimer's disease. J. Neurosci. 19, 1959–1964 (1999)). Therefore, atransgenic mouse is preferred in which neurofibrillary tangles exist ina state where they are bound to RNA since it resembles theneurofibrillary tangles in humans.

The nucleic acids in neurons can be subjected to specificcounterstaining with propidium iodide (PI). The levels of nucleic acidsare proportional to the density of stain. Therefore, if a pretreatmentwith RNase decreases the staining of cytoplasm with PI, the existenceand localization of nucleic acids can be confirmed by comparing thedensity before and after the pretreatment with RNase. Further, comparingthe localization of mutant tau aggregations with the localization of RNAby electron microscope, the binding of RNA and tau aggregations in NFTcan be confirmed.

<5> Screening and Producing Methods of Agent for TreatingNeurodegenerative Disorder

The transgenic mouse of the present invention can be used as a preclinicmodel of a neurodegenerative disorder for development of therapeuticagents for human neurodegenerative disorder.

Therefore, the present invention provides a method for screening for anagent for treating a neurodegenerative disorder by using the transgenicmouse of the present invention. The screening method of the presentinvention comprises administering a candidate substance to thetransgenic mouse of the present invention, or contacting a candidatesubstance with a tissue or a cell from brain of the transgenic mouse ofthe present invention, and determining an effect of the candidatesubstance on a property associated with the neurodegenerative disorderin the transgenic mouse or the tissue or the cell thereof.

The method of administering the candidate substance to the transgenicmouse is not particularly limited, and may be a usual method used formouse. As to the administration period, the administration is preferablyperformed from prior to apperance of neurofibrillary tangles to a timewhen neurofibrillary tangles usually appear in the transgenic mouse towhich the candidate substance is not administered. The method ofdetermining the effect of the candidate substance on the propertyassociated with the neurodegenerative disorder in the transgenic mouseis not particularly limited either. An example thereof is a method ofperforming a pathological test of brain.

The tissue or the cell from brain of the transgenic mouse includesisolated tissues and cells and cultured materials therefrom. The methodof contacting the candidate substance with the tissue or the cell is notparticularly limited either. An example thereof is a method ofdetermining whether tau protein insoluble even in the presence of SDSappears or not, by Western blotting.

It is preferred that the first screening is performed by contacting acandidate substance with a cell isolated from the transgenic mouse ofthe present invention, and then the second screening is performed byadministering the candidate substance selected by the first screening tothe transgenic mouse of the present invention. Specifically, the firstscreening is performed by simultaneously administering β-amyloid and acandidate substance to primary cultured cells of hippocampus from thetransgenic mouse and determining tau protein insoluble even in thepresence of SDS appears or not, by Western blotting, and the secondscreening is performed by administering the substance having an effectin the first screening to the transgenic mouse (for example, 10 months)and performing a pathological test to confirm an effect in theindividual level.

The screening method of the present invention can be used for productionof an agent for treating a neurodegenerative disorder. Namely, an agentfor treating a neurodegenerative disorder can be produced by preparing apharmaceutical composition comprising the substance selected by thescreening method of the present invention. The preparation ofpharmaceutical composition may be performed according to usual methodsdepending on kinds of the selected substance, forms of the preparationand the like.

EXAMPLE

The present invention will be described in more detail by an example.However, the present invention should not be limited thereto.

Example 1

<1> Generation of Transgenic Mice Expressing V337M Human Tau

FIG. 1 shows a map of a structure of a DNA encoding a mutant human tauused to generate a transgenic mouse of the present invention. Thisstructure contained a cytomegalovirus (CMV) enhancer, a PDGF-β promoter,and a human tau cDNA encoding mutation of from Val to Met at theposition of codon 337. Epitope tags (myc and FLAG) were attached to theN-terminal terminal and C-terminal thereof. The DNA encoding V337M humantau with myc and FLAG epitope tags was prepared by PCR-basedsite-specific mutation using a DNA having the nucleotide sequence ofGenBank/EMBL/DDBJ accession No. 03778 as a template. Specifically, thefollowing three sets of PCR primers were used.

-   (1) Sense: SEQ ID NO: 3    -   Antisense: SEQ ID NO: 4-   (2) Sense: SEQ ID NO: 5    -   Antisense: SEQ ID NO: 6-   (3) Sense: SEQ ID NO: 7    -   Antisense: SEQ ID NO: 8

The entire sequence of the cloned cDNA (1.4 kb) was confirmed by an ABIPRISM 377 DNA Sequencer. This CDNA was inserted into the XhoI and NotIsites of a PDGF-β chain expression vector prepared by inserting a clonedPDGF-β chain promoter portion into the CMV promoter portion of pCIneovector. A 4.3 kb BglII-NaeI fragment containing PDGF-p promoter, V337Mhuman tau cDNA and a 3′-untranslated sequence was used as a transgene togenerate a mutant tau transgenic mouse (Tg) on a B6SJL background.Microinjection of the transgene and generation of transgenic mice wereperformed in DNX Transgenic Sciences (Cranbury, N.J., U.S.A.). As aresult of the transplantation of 200 zygotes into which the transgenewas introduced into foster mothers, 70 infants were obtained. Three Tgmice among them were identified by Southern blot analysis of tail DNAusing ³²P-labeled human tau probe. Tg mouse lines established from thethree mice were named Tg216, Tg214 and Tg212, respectively.

<2> Western Blot Analysis of Tau Expression

To estimate tau expression and solubility in different buffers, brainsof transgenic mice and non-transgenic littermates were carefullyextracted after euthanisation with anaesthesia. Each brain was carefullyparted midsagittally. The right hemisphere was used forimmunohistochemistry and the left hemisphere was used for Western blotanalysis. Tissue was homogenized in 400 μl of RAB (0.1 M MES, 1 mM EGTA,0.5 mM MgSO₄, 0.75 M NaCl, 0.02 M NaF, and 1 mM phenylmethylsulfonylfluoride) and centrifuged at 50,000×g for 40 minutes at 4° C. in anOptima TL ultracentrifuge (Beckman). The pellet was re-homogenized with1 M sucrose/RAB, and centrifuged at 50,000 ×g for 20 minutes at 4° C.The resulting pellet was homogenized in 400 μl of RIPA buffer (50mMTris, 150 mM NaCl, 1% NP40, 5 mM EDTA, 0.5% sodium deoxycholate, and0.1% SDS, pH 8.0) and centrifuged at 100,000×g for 20 minutes at 4° C.The RIPA-insoluble pellet was solubilized in 70% formic acid (FA) andreconstituted in 50 μl Laemli SDS/PAGE sample buffer afterlyophilization. Protein concentration of each fraction was determined byCoomassie Brilliant Blue (CBB) dye (Nacalai). The samples were separatedby SDS/PAGE (8% acrylamide gel). The separated protein was blotted ontoImmobilon-P membrane (Millipore). After blocking the membrane with PBScontaining 10% skimmed milk solution and 0.1% Tween-20, the membrane wasincubated with anti-tau antibody JM diluted in PBS containing 5% skimmedmilk and 0.05% Tween-20 (×10000). The membranes were developed usingHRP-labeled anti-rabbit IgG polyclonal antibody. Chemiluminescentdetection (ECL, Amersham) was used for visualization.

<3> Preparation of Antibodies

The following antibodies were used:

-   (1) Mouse monoclonal anti-myc (clone 9E10; BabCO, Calif., U.S.A.)-   (2) Rabbit polyclonal anti-myc (MLC, Japan)-   (3) Phosphorylation-independent rabbit polyclonal anti-tau JM    (Takashima, A. et al., Presenilin 1 associates with glycogen    synthase kinase-3β and its substrate tau. Proc. Natl. Acad. Sci. USA    95, 9637–9641 (1998); Jansen, Belgium)-   (4) Rabbit polyclonal anti-ubiquitin (DAKO, Calif., USA)-   (5) Phosphorylation-dependent rabbit monoclonal anti-tau antibody    AT8 (Biernat, J. et al. The switch of tau protein to an    Alzheimer-like state includes the phosphorylation of two    serine-proline motifs upstream of the microtubule binding region.    EMBO J. 11, 1593–1597 (1997); it recognizes the phosphorylated tau    at Ser202 and Ser205; Innogenetics)-   (6) Phosphorylation-dependent rabbit polyclonal anti-tau antibody    PS199 (Michel, G. et al. Characterisation of tau phosphorylation in    glycogen synthase kinase-3β and cyclin dependent kinase-5 activator    (p23) transfected cells. Biochimica et Biophysica Acta 1380, 177–182    (1998); it recognizes phosphorylation of tau at Ser199; generously    donated by Ishiguro K., Mitsubishi Kasei Institute of Life Science)-   (7) Mouse monoclonal anti-tau antibody Alz50(Lee, V. M., Balin, B.    J., Otvos, L. Jr. & Trojanowski, J. Q. A68: a major subunit of    paired helical filaments and derivatized forms of normal Tau.    Science 251, 675–678 (1991); it recognizes the conformational    epitope found in paired helical filaments; generously donated by P.    Davies, Einstein University).    <4> Immunohistochemical and Histochemical Studies

Brains were immersion-fixed with 10% buffered formalin, andparaffin-embedded sections (2 to 10 μm) were prepared for either lightand confocal microscopic observation or electron microscopic observation(see below). Deparaffinized sections were treated in either 0.1% TritonX-100 in PBS for 20 minutes or Target Retrieval Solution (DAKO, Calif.,USA). Anti-myc, JM, PS199, AT8, Alz50, and anti-ubiquitin were used asprimary antibodies. Incubation in primary antibody solution occurredovernight at 4° C. Sections (2 μm) intended for light microscopicobservations were incubated with mouse monoclonal anti-myc antibody asthe primary antibody and goat anti-mouse IgG antibody conjugated byalkaline phosphatase as the secondary antibody. Visualization wasaccomplished with the HistoMark RED kit (KPL MD, USA). Sections intendedfor confocal laser microscopic observations were incubated with eitheranti-mouse Ig, labeled by Alexa488/568 (Molecular Probe), or anti-rabbitIg, labeled by Alexa 488/568, as the secondary antibody. For nuclearcounterstaining, PI or TO-PRO-3 dye (Molecular Probe) was used aftertreatment with RNase. PI recognizes nucleic acid. The sections werescanned with a Radiance 2000 KR3 (BIO-RAD, UK). Several sections werereacted in 0.05% Thioflavin S for 5 minutes and similarly scanned. 3 or4 sections were stained with Congo red after confocal laser microscopicobservation, and were then examined by light microscope using crossedpolarizing filters (NIKON).

<5> Studies on Microtubules

After the immunohistochemical and histochemical studies, an adjacentseries of paraffin-embedded sections (10 μm) were prepared for electronmicroscopic examination. Deparaffinized sections were hydrated andpostfixed in 1% osmium tetroxide, dehydrated in various grades ofethanol, infiltrated with propylene oxide, and embedded in Araldite(trademark of TAAB). Ultrathin sections were cut with an ultramicrotome,stained with uranyl acetate and lead citrate, and examined with aJEM-1200EX transmission electron microscope at 80 kV.

<6> Evaluation Results

Evaluation results are shown below.

Introduction of mutant human tau DNA into mice was confirmed by Southernblot analysis. Namely, DNA fragments from Tg216 (Lane 5), Tg214 (Lane6), and Tg212 (lane 7) line transgenic mice were analyzed by Southernblotting using XhoI- and NotI-digested tau DNA as the probe (FIG. 2). Ascontrols, mutant human tau DNAs (Transgene) before introduction to thetransgenic mice (Lanes 2 to 4 and 8) were also electrophoresed. Theresults revealed mutant human tau DNA was introduced in all thetransgenic mice.

The expression of mutant human tau (V337M) in the brains of transgenicmice (Tg) was confirmed by Western blot analysis (FIG. 3( a)). Namely,RAB fraction proteins (20 μg) from the brains of mice were each suppliedon 8% acrylamide gel, separated by SDS electrophoresis, and transferredto a membrane. Then, analysis was made using an anti-myc antibody, thatspecifically recognizes mutant human tau, as the probe and mutant humantau (V337M) was detected at the position of 70 kD. As a result, for allthe transgenic mice (Lane 3, Tg212; Lane 4, Tg214; and Lane 5, Tg216),expression of mutant human tau protein was confirmed. On the other hand,in the non-transgenic mice, mutant human tau was not detected (Lane 1,nontransgenic littermate of Tg214; and Lane 2, nontransgenic littermateof Tg21).

FIG. 3( b) shows the results obtained by probing anti-tau antibody, JM,which recognizes both endogenous and mutant human tau by Westernblotting. Lanes 1 to 5 are given same assignments as in FIG. 3( a). Boththe transgenic and non-transgenic mice showed similar electrophoreticpatterns.

Then, histological observation of brain tissues of transgenic mice wasperformed by staining the brain tissue sections and performingmicroscopic observation. Namely, Tg214 and Tg216 sections (2 μm)obtained from 10 month old mice were stained with mouse monoclonalanti-myc antibody as a primary antibody and alkaline phosphatase boundanti-mouse IgG as a secondary antibody, and then observed under lightmicroscope. FIG. 4( a) shows the cerebral cortex of Tg216. Expression ofmutant human tau in the cerebral cortex was confirmed. FIG. 4( b) showsthe hippocampus of Tg214. Similarly, expression of mutant human tau inthe hippocampus was confirmed (scale bar in (a) and (b); 50 μm).

As is apparent from the results shown in FIGS. 2, 3 and 4, mutant humantau DNA (V337M) was introduced into the transgenic mice (Tg212, Tg214,and Tg216) and expression of the mutant human tau DNA (V337M) in thebrain tissues of the mice was confirmed.

Adjacent series of sections of hippocampal region of Tg214 (10 monthsold) were specifically stained with the various antibodies set forthbelow and histochemical characteristics of the neurons of the transgenicmice were observed. The transgenic mice tissue sections werespecifically immunostained with mouse monoclonal anti-myc antibody andphosphorylation dependent rabbit polyclonal anti-tau antibody PS199. Thetissue sections were also specifically immunostained withphosphorylation dependent mouse monoclonal antibody AT8 and rabbitpolyclonal anti-ubiquitin antibody. Further, the sections were alsospecifically immunostained with rabbit polyclonal anti-myc antibody andmouse monoclonal antibody Alz50, an antibody specific to theconformational epitope found in PHF.

FIG. 5( a) to (c) are light microscopic photographs after staining mousebrain tissue sections (Tg214 (10 months old)) with Congo red. In thetissue of hippocampus of Tg214 (10 months old) (FIG. 5( a)), NFT wasobserved while in the littermate of Tg214, which is nontransgenic, noNFT was observed (FIG. 5( b)).

In the cerebral cortex of transgenic mice (FIG. 5( c)), NFT similar tothat in the Alzheimer patient (FIG. 5( d)) was observed (scale bar: 10μm). The existence of NFT was confirmed more significantly when observedunder a polarizing microscope. Both the neurons of transgenic mice andNFT of Alzheimer patient showed clear yellow filamentous structuresunder a crossed polarizing filter.

From the results in FIG. 5, it is apparent that specific immunostainingof transgenic mouse brain sections and their histological analysis bymicroscopic observation revealed that human neurodegenerative disorderspresent characteristic neurofibrillary tangles.

Further, electron microscopic observation of ultrathin sections of atissue of Tg214 mouse hippocampal neurons revealed a bundle of linearmicrotubules in neurons with irregularly shaped nuclear membranes. As aresult of further observation at greater magnifications, the diameter ofthe bundle of linear fibrils was measured to be 15 to 18 nm. Thus,electron microscopic observation indicated that linear microtubulesoccurred at the portion corresponding to PHF associated withneurofibrillary tangles (NFT) in the neurons obtained from transgenicmice, which suggests formation of neurofibrillary tangles.

When PI staining was performed after pretreatment with RNase, PI stainedonly the nuclei. On the other hand, when PI staining was performedwithout pretreatment with RNase, both the nuclei and the locations wherethe neurofibrillary tangles were confirmed were stained.

In the neurofibrillary tangles occurring in human brain with Alzheimer'sdisease, it was confirmed that the NFT existed in a state bound to RNA.This suggests that the transgenic mice exhibit neurofibrillary tanglessimilar to those of humans.

Furthermore, the transgenic mouse of the present application showedabnormality in behavior such as antianxiety, which is presumed to besimilar to the behavior of progressed Alzheimer patients.

Example 2

Except that R406W (arginine at 406 is substituted with tryptophan)humantau CDNA is used instead of V337M human tau cDNA, a transgenic mouseexpressing R406W human tau was generated in the same way as in Example1.

Analysis of brain sections in the same way as in Example 1<2> to <4>revealed that neurofibrillary tangles as in the V337M mouse wereobserved in hippocampus of the R406W mouse.

1. A transgenic mouse whose genome comprises a DNA sequence encoding ahuman tau protein operably linked to a promoter, wherein in said humantau protein has a mutation of valine at residue 337 of SEQ ID NO: 9 tomethionine, or a mutation of arginine at residue 406 of SEQ ID NO: 9 totryptophan, or both of said mutations, wherein the mouse brain exhibitsneurofibrillary tangles.
 2. The transgenic mouse according to claim 1,wherein the neurofibrillary tangles are positive in immunoreactionsusing an antibody against a phosphorylated tau protein, an antibodyagainst ubiquitin and an antibody against paired-helical filaments, andbirefringency is shown after staining with Congo red.
 3. The transgenicmouse according to claim 1, wherein the mouse shows abnormal behavior.4. The transgenic mouse according to claim 1, wherein the human tauprotein is expressed under control of platelet-derived growth factor βchain promoter.
 5. A method for screening for an agent for treatingneurofibrillary tangles, comprising administering a candidate substanceto the transgenic mouse of claim 1, or contacting a candidate substancewith brain tissue or a neuronal cell of said transgenic mouse, anddetermining the effect of the candidate substance on tau phosphorylationor tangle formation.
 6. The transgenic mouse according to claim 1,wherein the human tau protein is phosphorylated at serine residue 202and 205 of SEQ ID NO:
 9. 7. The transgenic mouse according to claim 1,wherein the human tau protein has the mutation of valine AT residue 337of SEQ ID NO: 9 to methionine.
 8. The transgenic mouse according toclaim 1, wherein the human tau protein has the mutation of arginine atresidue 406 of SEQ ID NO: 9 to tryptophan.
 9. The transgenic mouseaccording to claim 1, wherein the human tau protein has the mutation ofvaline at residue 337 of SEQ ID NO: 9 to methionine and the mutation ofarginine at residue 406 of SEQ ID NO: 9 to tryptophan.
 10. Thetransgenic mouse according to claim 7, wherein the human tau protein isexpressed under control of platelet-derived growth factor β chainpromoter.